Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test27settembre

From 2010.igem.org

Revision as of 11:16, 26 October 2010 by Chiara (Talk | contribs)

SEPTEMBER, 27TH



PLATE


UNIPV Pavia piastra27settembre.png


EXPERIMENT DESCRIPTION

Motivation

This experiment was performed to check bacterial growth and GFP rate synthesis of the following constructs in order to verify right protein folding.

Methods

Inoculum (into 5 ml LB+Amp) from glycerol stock of:

  • I47
  • I48
  • I49
  • <partinfo>BBa_K173000</partinfo> (positive control)
  • <partinfo>BBa_B0031</partinfo> (negative control)

They were let grow ON at +37°C, 220 rpm.

The following day cultures were diluted 1:100 and let grow again for about five hours at +37°C, 220 rpm.

Optical density (O.D.) of each cell culture was than measured with TECAN Infinte F200. Samples were diluted in order to obtain the same O.D. equal to 0,02.

Than we performed a 21 hours' experiment with measurements of absorbance and green fluorescence every five minutes with TECAN Infinite F200. Each value shown is the mean of three measurements, from GFP data that of a non-fluorescent culture (negative control) was subtracted; cultures were shaken for 15 seconds every five minutes.

RESULTS

Raw growth curve
CultureDoubling time [min.]
<partinfo>BBa_K173000</partinfo>77
I4774
I4875
I4976
<partinfo>BBa_B0031</partinfo>69
Raw GFP curve
Mean of (dGFP/dt)/O.D.600 (under the hypothesis that half-life of GFP is longer than experiment observation)

All cell cultures showed a similar growth curve and doubling time was computed as described here in order to have informations about the burden due to synthesis of such fusion proteins. It's possible to see that all doubling time are very similar; it's possible to assert that the expression of these BioBrick parts doesn't cause abnormal stress to the cells.

In GFP curve it's possible to appreciate that in I47, I48, I49 GFP accumulation it's very similar and it's significantly different from that of negative control <partinfo>BBa_B0031</partinfo>. These results show the right folding of the green fluorescent protein assembled downstream of the genetic circuit.

A mean protein synthesis rate was also computed over the growth exponential phase, showing again an appreciable GFP production rate that is about a half of the positive control.



Tecan Test